Understanding the mechanism by which individual cells choose between alternative developmental pathways is critical if normal development and its failures are to understood. Sex differentiation in Drosophila melanogaster offers an excellent system in which to approach these problems. The primary inducing signal is known and separation of tissues following alternative pathways is achieved by the separation of individuals. In addition, the genes involved in regulating the process have been identified genetically and isolated molecularly, and the outlines of the interactions between these genes have been determined. Multiple aspects of regulation within this hierarchy will be studied at the molecular level. The transformer (tra) gene is regulated by sex-specific alternative RNA splicing. Tests of models for this regulation and mapping of the necessary sites will be carried out both in vivo and in vitro. Factors which interact with the regulated intron will be identified and used to reconstitute a sex-specific splicing system. tra is also involved, in a process requiring the product of the transformer-2 (tra-2) gene, in the sex-specific alternative processing of RNA from the doublesex (dsx) gene. The tissue and subcellular distribution of the tra protein will be determined and interaction with other proteins, specifically with tra-2, will be examined. Sites of interaction between the tra protein and dsx RNA will be mapped. Parts of the tra protein necessary for interaction with dsx RNA or with other proteins will be identified by mutation and mutations will be tested for their in vivo and in vitro effects. Genes which function downstream of the sex determination hierarchy are involved in the production of sex-specific structures and the elaboration of sex-specific behaviors. They also represent genes which may be integrating information from both the sex differentiation hierarchy and from the set of genes controlling segment and positional identity. In order to fully understand the mechanism of action of the sex differentiation hierarchy, and to map out any additional branches of the hierarchy, it is critical to identify downstream targets for the action of these genes. Genes expressed, at multiple different developmental stages, in a sex-specific manner will be isolated. The expression of these genes will be characterized with regard to their control by various different elements of the sex hierarchy as a way of distinguishing genes involved in dosage compensation, germ line sex determination, sexual differentiation and reproductive behavior. These genes will be characterized with regard to genetic position, temporal and spatial patterns of expression, and mechanism of regulation.